Our overall goal is to understand how the breakdown of normal self- tolerance mechanisms leads to systemic autoimmune disease. Transgenic (Tgic) mouse systems developed by several groups including our own have demonstrated that self-reactive B cells can be regulated by a remarkably diverse set of mechanisms, including deletion, anergy and receptor edition. We have shown that B cells expressing a disease-associated Rheumatoid Factor (RF, anti-IgG2a), autoAb are not subject to any of these known regulatory mechanisms in normal mice, a state termed clonal ignorance. Such ignorant lymphocytes may be the precursors of truly pathogenic autoimmune responses. Thus, autoimmune mice differ from normal mice in regulating autoreactive B cells at stages beyond deletion and anergy of newly developed B cells. There are many Ag receptor dependent stages of B cell development--notably development and maintenance of memory B cells--where regulatory checkpoints could exist but have been little explored. By focusing on these later stages of B cell development, we plan to address the fundamental questions: How do normal mice prevent the development of autoreactive B cell clones, whereas autoimmune mice promote development, and how is this autoimmunity sustained? To address this, we will test two hypothesis. The first is that autoAb-secreting clones arise from low affinity precursors that are not censored by anergy or deletion in normal mice, but in autoimmune-prone mice, these clones undergo Ag-driven affinity maturation and memory B cell differentiation. This will be tested in Aim 1 where we will use our AM14 Tgic system and V region "knockin" versions of it to compare RF B cell memory development in both normal and autoimmune mice in the presence and absence of autoAg. The second hypothesis, suggested by new data from our high affinity RF Tgic system could explain chronicity. We hypothesize that once autoreactive B cells secrete sufficient autoAb for a given specificity, the autoAg can no longer delete or anergize B cells specific for that autoAg, because the presence of the autoAb removes or block the autoAg. This will be tested in Aim 2 where we will seek direct evidence that a self-amplifying circuit can propagate autoAb production in both Tgic and non-Tgic mice. This work could elucidate a novel mechanism by which autoimmunity, once induced, may be propagate indefinitely, an important feature of human SLE. To determine where B cell regulation fails in systemic autoimmunity is critical, as it would permit molecular studies directed at the cellular mechanism and would provide specific therapeutic targets. This is the ongoing focus of our lab and of this proposal.